Conformational cycling in β-phosphoglucomutase catalysis:: Reorientation of the β-D-glucose 1,6-(bis) phosphate intermediate
Conformational cycling in β-phosphoglucomutase catalysis:: Reorientation of the β-D-glucose 1,6-(bis) phosphate intermediate
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DOI：
10.1021/bi060136v
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发表时间：
2006-06-27
期刊：
影响因子：
2.9
通讯作者：
Dunaway-Mariano, Debra
中科院分区：
文献类型：
--
作者：
Dai, Jianying;Wang, Liangbing;Dunaway-Mariano, Debra
活化的乳酸乳球菌β-磷酸葡萄糖变位酶（β PGM）催化源自麦芽糖的β-D-葡萄糖1-磷酸（β G1 P）转化为β-D-葡萄糖6-磷酸（G6 P）。活化需要Mg 2+结合和活性位点残基Asp 8的磷酸化。初始速度技术用于确定稳态动力学常数k（cat）= 177 +/- 9 s（-1），底物β G1 P的K-m = 49 +/- 4 μ M，活化剂β-D-葡萄糖1，6-二磷酸（β G1，6 bisP）的K-m = 6.5 +/- 0.7 μ M。在用过量β PGM（40 μ M）和限制性[C-14] β G1 P（5 μ M）和β G1，6 bisP（5 μ M）进行的单次转换反应中观察到的[C-14] β G1，6 bisP（12%，近似0.1 s）的瞬时积累支持β G1，6 bisP作为G1 P转化为G6 P的反应中间体的作用。进行了[C-14] β G1，6 bisP与过量β PGM的单周转反应，以证明磷酰基转移而不是配体结合是限速的，并表明β G1，6 bisP以两种不同的方向与活性位点结合（一个定位C（1）磷酰基以与Asp 8反应，而另一个取向定位C（6）磷酰基以与Asp 8）反应，具有大致相同的效率。用β PGM、[C-14] β G1 P和未标记的β G1，6 bisP进行的单周转反应证明在催化循环期间标记完全交换为β G1，6 bisP。因此，完成催化循环所需的β G1，6 bisP中间体的重新取向通过扩散到溶剂中，然后以相反的取向结合而发生。已发表的β G1 P的X射线结构表明，β G1，6 bisP的重定向和磷酰基转移是通过酶在活性位点开放和封闭形式之间的构象循环通过帽结构域运动发生的。最后，检查β G1，6 bisP与β G1 P加G6 P的平衡比率，以证明β PGM γ-磷酸的显著稳定性。
Activated Lactococcus lactis beta-phosphoglucomutase (beta PGM) catalyzes the conversion of beta-D-glucose 1-phosphate (beta G1P) derived from maltose to beta-D-glucose 6-phosphate (G6P). Activation requires Mg2+ binding and phosphorylation of the active site residue Asp8. Initial velocity techniques were used to define the steady-state kinetic constants k(cat) = 177 +/- 9 s(-1), K-m = 49 +/- 4 mu M for the substrate, beta G1P and K-m = 6.5 +/- 0.7 mu M for the activator beta-D-glucose 1,6-bisphosphate (beta G1,6bisP). The observed transient accumulation of [C-14]beta G1,6bisP (12% at similar to 0.1 s) in the single turnover reaction carried out with excess beta PGM (40 mu M) and limiting [C-14]beta G1P (5 mu M) and beta G1,6bisP (5 mu M) supported the role of beta G1,6bisP as a reaction intermediate in the conversion of the, G1P to G6P. Single turnover reactions of [C-14]beta G1,6bisP with excess, beta PGM were carried out to demonstrate that phosphoryl transfer rather than ligand binding is rate-limiting and to show that the beta G1,6bisP binds to the active site in two different orientations (one positioning the C(1) phosphoryl group for reaction with Asp8, and the other orientation positioning the C(6) phosphoryl group for reaction with Asp8) with roughly the same efficiency. Single turnover reactions carried out with beta PGM, [C-14]beta G1P, and unlabeled beta G1,6bisP demonstrated complete exchange of label to the beta G1,6bisP during the catalytic cycle. Thus, the reorientation of the beta G1,6bisP intermediate that is required to complete the catalytic cycle occurs by diffusion into solvent followed by binding in the opposite orientation. Published X-ray structures of beta G1P suggest that the reorientation and phosphoryl transfer from beta G1,6bisP occur by conformational cycling of the enzyme between the active site open and closed forms via cap domain movement. Last, the equilibrium ratio of beta G1,6bisP to beta G1P plus G6P was examined to evidence a significant stabilization of beta PGM aspartyl phosphate.